Antibacterial Activity of Mahajak Remedy and Plant Ingredients
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P-ISSN 2586-9000 E-ISSN 2586-9027 Homepage : https://tci-thaijo.org/index.php/SciTechAsia Science & Technology Asia Vol.25 No.3 July - September 2020 Page: [78-86] Original research article Antibacterial Activity of Mahajak Remedy and Plant Ingredients Phadungnaree Saenmuang1, Sumalee Panthong 1,2, Arunporn Intharat1,2,* 1Department of Applied Thai Traditional Medicine, Faculty of Medicine, Thammasat University, Pathum Thani 12120, Thailand 2Center of Excellence in Applied Thai Traditional Medicine Research, Faculty of Medicine, Thammasat University, Pathum Thani 12120, Thailand Received 28 October 2019; Received in revised form 20 June 2020 Accepted 26 June 2020; Available online 21 September 2020 ABSTRACT Mahajak (MH) oil is a remedy in Tamra-Osodpranarai. It is used for relieving pain, and skin infection as a Thai traditional medicine. This study investigated the efficacy of the MH remedy and plant ingredient extract on antibacterial activities. The MH remedy was macerated with sesame oil, 95% ethanol, hexane, and its plant ingredients were macerated with 95% ethanol. Then, all extracts were concentrated by rotary evaporator. The extracts were investigated on antibacterial activity against Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 9027, and Bacillus subtilis ATCC 6633 By disc diffusion method, minimum inhiBitory concentration (MIC) and minimum Bactericidal concentration (MBC). The results show that the 95% ethanol extract of MH remedy was the best extract of antibacterial testing. Using the disc containing 10 µL (concentrate 500 mg/mL of the extracts), the inhibition zone was observed against S. aureus (9.67±0.33 mm). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of 2.50 mg/mL were oBtained for residue hexane. Hexane extracts showed MIC and MBC of S.aureus and B.subtilis with similar values to 95% ethanol. However, all of the tests had no inhibitory activity against P.aeruginosa. In conclusion, this study demonstrated that 95% ethanol extracts of the MH remedy have antiBacterial activity and may Be a potential treatment in cases of skin infection. Keywords: AntiBacterial activities; Mahajak oil; Tamra-osodpranarai *Corresponding author: [email protected] doi: 10.14456/scitechasia.2020.37 P. Saenmuang et al. | Science & Technology Asia | Vol.25 No.3 July – September 2020 1. Introduction However, there is no scientific report Bacterial skin infections are caused by on the antibacterial activity of this remedy. many types of Bacteria such as Thus, the oBjective of this study was to Staphylococcus aureus, Pseudomonas investigate the antibacterial activity of MH aeruginosa, and Bacillus subtilis [1]. Skin extracts involved in skin infection and infection includes cellulitis, erysipelas, inflammation. impetigo, folliculitis, furuncles and carbuncles, which can be classified by the 2. Materials and methods depth and skin compartment [2, 3]. The 2.1 Plant materials World Health Organization (WHO) has The MH remedy, including Nigella estimated that 56.9 million people died sativa L, Lepidium sativum L, Cuminum worldwide in 2016, and infectious diseases cyminum L, Foeniculum vulgare Miller, were one cause of death [4]. AntiBiotics, such Anethum graveolens L., and Piper as penicillin, clindamycin, and retrofractum Vahl., was obtained from a Thai cephalosporin, are widely used to treat skin herbal drug store in Bangkok. The infection [5]. However, Bacteria that still ingredients were confirmed by comparison survive on antibiotic treatment are called with authentic voucher specimens that were antibiotic-resistant. Antibiotic resistance has kept in the herbarium of Southern Center of become a global health problem. In 2018, the Thai Medicinal Plants, Faculty of Phar- National AntimicroBial Resistance maceutical Sciences, Prince of Songkla Surveillance Center in Thailand reported that University, Songkhla, Thailand. The herBar- antibiotic resistance was found in all parts of ium voucher specimen numBer is shown in Thailand, such as S. aureus resistant to (TaBle 1). Plant ingredients were washed methicillin [6]. At the present time, many with water to remove contamination and researchers are interested in an antimicro- dried at 50ºC for 72 hr. Each plant was bial agent from a natural product. Secondary ground to powder and weighed according to metaBolites from plants, such as tannins, the recipe of the MH remedy in the alkaloids, and quinones, can inhibit patho- Osodpranarai scripture. gens. Thus, many natural products have been investigated as antimicrobial agents and in 2.2 Preparation of crude extract the development of antimicrobial agents [7]. Plant ingredients of the MH remedy In Thailand, traditional Thai doctors have were extracted by the traditional method and used Thai traditional remedies that comBine conventional method. For the traditional many types of herBal plant as a drug for the method, plant ingredients were macerated in treatment of patients for a long time. sesame oil, while an organic solvent such as Mahajak (MH) is a traditional Thai remedy ethanol and hexane were used for the which has Been descriBed in the conventional extraction method. Osodphranarai scripture. It has long been 2.2.1 Maceration in sesame oil used to relieve pain and reduce skin Firstly, fresh peel of kaffir lime was inflammation. At present, the MH remedy is added into sesame oil (the ratio of 2:1) and still used By Thai Traditional doctors in heated at 150ºC for 25-30 min. Then, the LopBuri’s hospital to treat skin inflame- sesame oil was filtered, cooled down to room mation. The MH remedy consists of Nigella temperature, and used for maceration of plant sativa L., Lepidium sativum L., Cuminum ingredients. Plant powder ingredients were cyminum L., Foeniculum vulgare Miller., separated into two parts. The first part and Anethum graveolens L., Piper retrofractum second part of plant ingredients were Vahl., Citrus hystrix DC., and Sesamum macerated with a sesame oil volume of 600 indicum L. [8,9]. mL and 200 mL for 15 days to oBtain MH oil 79 P. Saenmuang et al. | Science & Technology Asia | Vol.25 No.3 July – September 2020 remedy extract (MH1) and MH oil 2.3.1 Loss on drying concentrate extract (MH2). The electronic moisture analyzer 2.2.2 Maceration in an organic was used for the analysis of loss on drying. solvent Each plant (2 g) was put into the moisture The crude powder was extracted By analyzer and heated to 105°C. The weight maceration with hexane and 95% ethanol for (W) of the dried sample was taken and three days and filtered through Whatman analyzed by using the following equation. filter paper no1. The maceration process was W start (g) − W dried repeated twice more with hexane and 95% %Loss on drying = × 100 ethanol. The hexane extract (MH3) and W start (g) ethanolic extract (MH4) were concentrated by rotary evaporator, vacuum dried for one 2.3.2 Ethanol-soluble extractive day, and stored at -20ºC until used. The value residues of MH1, MH2, and MH3 were Five grams of the powdered drug extracted by maceration with 95% ethanol was macerated with 100 mL of ethanol in a and used the same procedure as MH4 flask that was closed with foil for 24 hrs. extraction. Finally, we obtained MH (Oil Moreover, it was occasionally shaken over remedy) extract (MH5), residue of MH (Oil the first 6 hrs. Then, it was allowed to stand concentrate) extract (MH6), the residue of for 18 hrs. and filtered. After filtration, 25 hexane extract (MH7). mL of the filtrate was evaporated at 105°C to The yields of MH1- MH7 extracts dryness in a tare flat-bottomed shallow dish, were calculated as the percentage of yield by then dried and weighed. Percentage of using the following equation: ethanol-soluBle extractive value was calculated with the below formula Weight of dried extract (g) %Yield = × 100 W extract (g) Weight of dried crude power (g) %Extractive value = × 100 W dried plant material (g) 2.3 Quality control of plant ingredients [10] 2.3.3 Total ash All plant ingredients were tested for This method was used to investigate quality control including loss on drying, the physiological ash and non-physiological extractive value, total ash, and insoluble acid ash or inorganic compounds of plant ash by using the guidelines of the Thai material. HerBal Pharmacopoeia. Table 1. Plant materials of MH remedy. Voucher Part Weight Scientific Name Family Name Thai Name specimen formulation number Nigella sativa L. Ranunculaceae Thian-Dam Seed SKP 160 14 19 01 15 g Lepidium sativum L. Cruciferae Thian-Daeng Seed SKP 057 12 19 01 15 g Cuminum cyminum L. Umbelliferae Thian-Khao Seed SKP 199 03 03 01 15 g Foeniculum vulgare Miller subsp. Umbelliferae Thian- Khaoplueak Seed SKP 199 06 22 01 15 g Anethum graveolens L. Umbelliferae Thian- Tatakkataen Seed SKP 199 01 07 01 15 g Piper retrofractum Vahl. Piperaceae Dipli Fruit SKP 146 16 18 01 30 g 80 P. Saenmuang et al. | Science & Technology Asia | Vol.25 No.3 July – September 2020 Total ash was determined by bacterium was swaBbed on Mueller Hinton weighing 2 g into a pre-weighed cruciBle and agar (MHA). Air-dried discs were placed on burning in a muffle furnace at 450°C for 5 hr. the inoculated MHA surface. An antibiotic Then, the cruciBle was placed into the commercial disc of ampicillin 10 µg/disc was desiccator to cool down. Finally, crucible used as a positive control. Negative control with ash was weighed again. The percentage was a disc that contained 10 µL of 2% of total ash was calculated by using the DMSO. Plates were incuBated at 37ºC for 18- following equation. 24 hr. After incubation, the inhibition zone diameter was measured. These tests were W after burning (g) %Total ash = × 100 performed in triplicate. W before burning (g) 2.4.2 Determination of minimum inhibitory concentration (MIC) [12] 2.3.4 Acid insoluble ash The MIC value was determined by a The ash oBtained from the previous broth dilution method.